Process for the manufacture of compound epi-f



United States Patent Q PROCESS FOR THE MANUFACTURE OF COMPOUND EPI-F Emilio Testa, Milan, Italy, assignor to Lepetit S. p. A.,

Milan, Italy N Drawing. Application March 28, E57 Serial No. 649,003

4 Claims. (Cl; 195-51) The present invention relates to the preparation of A -pregnene-11a,17a,2l-triol-3,20-dione (Compound epi- F). -More particularly this invention is concerned with a new method for preparing Compound epi-F by the microbiological hydroxylation of Reichsteins Compound S, A -pregnene-l7a,21-diol-3,20-dione, using the fungus Coryneum cardiizale 952 (LCI) (ATCC 130631).

In patents and in literature, different methods for converting the Compound S of Reichstein to ll-oxygenated steroids and, in particular, to compound F and to Compound epi-F by the use of different organisms, have been already described. For instance, U. S. Patent 2,602,769 describes the use of Cunninghamella blakesleana (order Mucorales); a publication in J. Amer. Chem. Soc. 74, 2381 (1952) describes a process utilizing Sz'r'eptomyces fradz'ae; U. S. Patent 2,658,023 describes the use of a fungus from the genus Curvularia (order Moniliales), while a fungus of the genus Pestalotia is utilized by the process of U. S. Patent 2,721,163. The importance of the introduction of oxygen into the ll-position of steroids is well known to the chemists skilled in the steroid field. Particularly, by converting Compound S into its 11-hy droxyl derivative,in the case of the hydroxyl being sterically in 118-positio-n, hydrocortisone, i. e. Compound F, is obtained and, in the case of hydroxyl entering into the Ila-position, Compound epi-F is obtained, which may be easily converted into cortisone by chemical processes.

We have now found that Compound S may be con-- verted into Compound epi-F in a quantitative yield by using the microorganism Coryneum cardinale 952 (LCI).

This microorganism belongs to the family Melanconiaceae, order Melanconiales, class Fungi Imperfecti. In identical conditions we have tested other species of the same genus, but we have not been able to detect in the culture broths, by chromatography, any trace of Compound F or epi-F. On the other hand, Coryneum cardinale 952 (LCI) as stated above effects the conversion in a 100% yield and no noticeable amounts of steroidal by-products are found in the culture broths, as was to be expected in view of the quantitative yield. 7

The strain Coryneum cardinals has been filed to the Italian Cryptogamic Laboratory of the Board of Agriculture and Forests at Pavia and it is listed with the No. 952. A culture has also been deposited with the American Type Culture Collection in Washington, D. C., where it has received the designation ATCC 13063T.

The process of the present invention may be applied both to Compound S and to 21-carboxylic acid esters of Compound S, such as 21-acetate, 2l-propionate, and so forth, in any case Compound epi-F is obtained in about a quantitative yield. The composition of the nutrient medium, the temperature, etc., must be properly adjusted according to the nature of the starting ester.

The lla-hydroxylation for converting Compound S to Compound epi-F is generally carried out by processes which are Well known to microbiologists. Coryneum cardinale 952, for instance, is grown in a usual nutrient medium with agar containing proteins and carbohydrates,

starch, glucose and dextrins.

The pH of the vegetative medium may also vary between broad limits from 3.5 to 8.0; however the best results have been obtained at pH from 5.0 to 6.0. The temperature may range between 10 to 40 C. but the best temperatures for spore production range between 24 and 30 C. After having obtained spo-rulation from a culture of Coryneum cardinale 952 in the vegetative medium, by a 2-10 days cultivation, the medium containing the spores is diluted and repeatedly washed with sterile water. The obtained spore suspension is inocualted in the fermentation medium which has nearly the same composition of vegetative medium and in which the vegetative mycelium forms in a time ranging from 12 to 48 hours. From this culture containing the vegetative mycelium, inocula are transferred into Erlenmeyer flasks containing fermentative medium and well shaken. After having obtained a good growth i. e., after 624 hours, Compound S dissolved in a lower aliphatic alcohol is added to the culture medium until a concentration from 1 to'5% of Compound S in culture medium, and preferably 2% is obtained. The fermentation medium is shaken until complete conversion of Compound S to Compound epi-F, which requires atime ranging between 10 and 24 hours. The conversion rate of Compound S into Compound F is determined bypaper chromatography repeated, for instance, every hour. Fermentation is stopped when the chromatography shows that no more Compound S but only Compound epi-F is present in the medium.

Instead of taking out the inoculum from the vegetative medium in which the spores have been grown, the fermentation medium may be directly inoculated with the spore suspension, whereby high yields of Compound epi-F are still obtained, although in a longer time;

Compound epi-F is ultimately extracted from the fermentation medium containing the mycelium, by a water immiscible solvent in which Compound epi-F is soluble, as for instance chloroform, carbon tetrachloride, methylene chloride, etc. The extracts are concentrated in vacuo to dryness and the residue is crystallized from a proper solvent,'as for instance acetone.

The following examples clearly illustrate the invention, although they are not intended as limiting the same.

Example A culture of Coryneum cardinale 952 (LCI) (ATCC 130631) is grown in an agar medium containing 1% peptone and 3% glucose for 3 days at 28 C. and at above spore suspension and the mixture is shaken in a rotative. shaker at revolutions/minute, at 30 C. for 24 hours. After this time to each flask a solution of 0.1 g. of Compound S in 5 ml. of methyl alcohol is added. Shaking is continued for 24 hours at 30. Chromatog raphy shows that conversion occurs at the following rates:

Time (hours) Percent conversion At the end of the fermentation the pH of the medium is 6.0. After 24 hours the material contained in the 10 V Patented Dec. 23, 1958 An inoculumof Coryneum cardinale 952 (L01) is prepared as described in Example 1. I

In 10 Erlemeyer flasks containing each 50 ml. of a termentation medium with 0.2% yeast extract, 1% glucose and 3% dextrins, 1 ml. of the spore suspension is inoculated, followed by'a solution of 0.1 g. of Compound S in 5' ml.'of ethyl alcohol and the mixture is shaken in a rotative' s'haker' at 28 C. for 48 hours, at 200 revolutions/minute. Chromatography shows that conversion occurs at the following rates:

Time (hours) Percent conversion Negligible 8 I I 2 12. 15

At the end of the fermentation the pH- of the medium is 5.7. After 48 hours the contents of the flasks are combined and extracted with chloroform. It is worked up asdescribed in Example 1, yielding 0.90 g. of Compound epi-F, M. P. 218-220 C.

Example 3 An inoculum ofCoryneum cardinale 9 52 (LCI) is prepared as described in Example l. Twenty five litres of a culture medium containingpeptone 0.8%, sucrose'3%, corn steep liquor 1% in-water are charged into a 100 liter fermentation tank. After sterilization the pH of the medium is about 6.0. 2% of inoculum is added and'the mixture iskept at 27 for 24 hours under stirring at-150 revolutions per minute while introducing sterile air.

Once the mycelium growth is complete, 25 g. of Comp'ound S dissolved in 300ml. of methyl alcohol, are added and the fermentation iscarriedoni until'complete conversion, i. e. for about 30'hours;the end pH is'5.4.

The mixture is thoroughly extracted with chloroform and the chloroform is evaporated todryness. The-residue is recrystallized from acetone, whereby a first crop of 18 g. of Compound epi-F of M. P. 2132l4 C., is obtained.

On acetone concentration a second crop of 6 g., M. P. 210214 C. is obtained.

By recrystallization from 'acetone the two combined crops yield 23 g. of Compound epi-F of M. P. 2l9-220 C.

Example "4 An inoculum of Coryneum cardinale 952 (LCI) is prepared as described in Example 1.

In 10 Erlenmeyer flasks, each containing ml. of a fermentation medium with 0.3 liver extract, 1% sucrose and 0.5% starch, 2 ml. of the spore suspension are inoculated followed by a solution of 0.2 g. of Compound S 21-acetate in 5 ml. of methyl alcohol and the mixture is shaken in a rotative shaker at 26 C. for 36 hours at 200 revolutions/ minute.

After 30 hours the conversion to compound isalready practically complete. Compound epi-F is isolated as indicated in the preceding examples, 1.75 g. are obtained. M. P. 218219 C. Using Compound S propionate as the starting material and fermenting for 48 hours at 29 an identical result is obtained.

I claim:

1. Process for preparing M-pregnene-l1a,17a,21-triol- 3,20-dione (Compound epi-F) starting from a steroid of of the group consisting of M-pregnene-17a,21diol-3,20- dione (Compound S) and its lower aliphatic carboxylic acid esters which comprises contacting said steroid with a strain of Coryneum cardinale 952 (LC!) (ATCC 13063T).

2. Process as described in claim 1, wherein Coryne'um cardinale 952 (LCI) is grown in a nutrient medium until mycelial growth'is obtained and the steroid is contacted with the mycelium.

3. Process for preparing A -pregnene-11a,l7a,21-triol- 3,20-dione starting from M-pregnene-17a,21-dio1-3,20-dione, substantially as described in claim 1.

4. Process for preparing A -pregnene-11a,17a,21-triol- 3,20-dione starting from asteroid of the group consisting of M-pregnene-17a,21-diol 3,20-dione and its lower aliphatic carboxylic acid esters, which comprises contacting said steroid in submerged aerobic conditions, with a strain of Coryneum cardinale 952 (LCI) (ATCC 13063T) at a temperature between 24 and 30 C. for a period of 12 to 48 hours.

Shull et al. Oct. 18, 1955 

1. PROCESS FOR PREPARING $4-PREGNENE-11A,17A,21-TRIOL3,20-DIONE (COMPOUND EPI-F) STARTING FROM A STERIOD OF OF THE GROUP CONSISTING OF $4-PREGNENE-17A,21 DIOL-3,20DIONE (COMPOUND S) AND ITS LOWER ALIPHATIC CARBOCYLIC ACID ESTERS WHICH COMPRISES CONTACTING SAID STEROID WITH A STRAIN OF CORYNEUM CARDINALE 952 (LCI) (ATCC 13063T). 